Human amine receptor

ABSTRACT

A Human amine receptor polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also provided are methods for detecting compounds which bind to and activate and bind to and inhibit such polypeptide and the use of compounds for treating diseases related to the under-expression and over-expression of the Human amine receptor of the present invention. Also disclosed are methods for detecting mutations in the nucleic acid sequence encoding the polypeptide and for detecting altered levels of the soluble form of the polypeptide.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention are human 7-transmembrane receptors and has been putativelyidentified as a human amine receptor. The invention also relates toinhibiting the action of such polypeptides.

It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., CAMP(Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins arereferred to as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the GPC receptors suchas those for adrenergic agents and dopamine (Kobilka, B. K., et al.,PNAS, 84:46-50 (1987); Kobilka, B. K., et al., Science, 233:650-656(1987); Bunzow, J. R., et al., Nature, 336:783-787 (3,988)), G-proteinsthemselves, effector proteins, e.g. phospholipase C, adenyl cyclase, andphosphodiesterase, and actuator proteins, e.g., protein kinase A andprotein kinase C (Simon, M. I., et al., Science, 252:802-8 (1991)).

For example, in one form of signal transduction, the effect of hormonebinding is activation of an enzyme, adenylate cyclase, inside the cell.Enzyme activation by hormones is dependent on the presence of thenucleotide GTP, and GTP also influences hormone binding. A G-proteinconnects the hormone receptors to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by hormone receptors. TheGTP-carrying form then binds to an activated adenylate cyclase.Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns theG-protein to its basal, inactive form. Thus, the G-protein serves a dualrole, as an intermediate that relays the signal from receptor toeffector, and as a clock that controls the duration of the signal.

The membrane protein gene superfamily of G-protein coupled receptors hasbeen characterized as having seven putative transmembrane domains. Thedomains are believed to represent transmembrane α-helices connected byextracellular or cytoplasmic loops. G-protein coupled receptors includea wide range of biologically active receptors, such as hormone, viral,growth factor and neuroreceptors.

G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc., Rev., 10:317-331 (1989)).Different G-protein α-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

The Human Amine Receptor of the present invention is a G-protein coupledreceptor. Neurosensory and neuromotor functions are carried out byneurotransmission. Neurotransmission is the conductance of a nerveimpulse from one neuron, called the presynaptic neuron, to anotherneuron, called the postsynaptic neuron, across the synaptic cleft.Transmission of the nerve impulse across the synaptic cleft involves thesecretion of neurotransmitter substances. The neurotransmitter ispackaged into vesicles in the presynaptic neuron and released into thesynaptic cleft to find its receptor at the postsynaptic neuron.Transmission of the nerve impulse is normally transient.

An essential property of synaptic transmission is the rapid terminationof action following neurotransmitter release. For manyneurotransmitters, including catecholamine, serotonin, and certain aminoacids (e.g., gamma-aminobutyric acid (GABA), glutamate and glycine),rapid termination of synaptic action is achieved by the uptake of theneurotransmitter into the presynaptic terminal and surrounding glialcells. This rapid re-accumulation of a neurotransmitter is the result ofre-uptake by the presynaptic terminals.

At presynaptic terminals, the various molecular structures for re-uptakeare highly specific for such neurotransmitters as choline and thebiogenic amines (low molecular weight neurotransmitter substances suchas dopamine, norepinephrine, epinephrine, serotonin and histamine).These molecular apparatuses are receptors which are termed transporters.These transporters move neurotransmitter substances from the synapticcleft back across the cell membrane of the presynaptic neuron into thecytoplasm of the presynaptic terminus and therefore terminate thefunction of these substances. Inhibition or stimulation ofneurotransmitter uptake provides a means for modulating the effects ofthe endogenous neurotransmitters.

The neurotransmitter substances are implicated in numerouspathophysiologies and treatments including, movement disorders,schizophrenia, drug addiction, anxiety, migraine headaches, epilepsy,myoclonus, spastic paralysis, muscle spasm, schizophrenia, cognitiveimpairment, depression, Parkinson's Disease and Alzheimer's Disease,among others.

Re-uptake of neurotransmitter substances by the transporters may besodium-dependent. For instance, the GABA transporter is a member of therecently described sodium-dependent neurotransmitter transporter genefamily. These transporters are transmembrane receptor complexes havingan extracellular portion, a transmembrane portion and an intracellularportion. A significant degree of homology exists in the transmembranedomains of the entire family of sodium-dependent neurotransmittertransporter proteins, with considerable stretches of identical aminoacids, while much less homology is apparent in the intracellular andextracellular loops connecting these domains. The extracellular loop inparticular seems to be unique for each transporter. This region maycontribute to substrate and/or inhibitor specificities.

The polypeptide of the present invention has been putatively identifiedas an amine receptor. This identification has been made as a result ofamino acid sequence homology to the rat amine receptor.

In accordance with one aspect of the present invention, there areprovided novel mature receptor polypeptides as well as biologicallyactive and diagnostically or therapeutically useful fragments, analogsand derivatives thereof. The receptor polypeptides of the presentinvention are of human origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding the receptorpolypeptides of the present invention, including mRNAs, DNAs, cDNAs,genomic DNA as well as antisense analogs thereof and biologically activeand diagnostically or therapeutically useful fragments thereof.

In accordance with a further aspect of the present invention, there areprovided processes for producing such receptor polypeptides byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing nucleic acid sequences encodingthe receptor polypeptides of the present invention, under conditionspromoting expression of said polypeptides and subsequent recovery ofsaid polypeptides.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such receptor polypeptides.

In accordance with another aspect of the present invention there areprovided methods of screening for compounds which bind to and activateor inhibit activation of the receptor polypeptides of the presentinvention.

In accordance with still another embodiment of the present inventionthere are provided processes of administering compounds to a host whichbind to and activate the receptor polypeptide of the present inventionfor the prevention and/or treatment of abnormal conditions resultingfrom under-expression of the amino receptor of the present invention.

In accordance with another aspect of the present invention there isprovided a method of administering the receptor polypeptides of thepresent invention via gene therapy to treat conditions related tounder-expression of the polypeptide or underexpression of a ligand tothe receptor polypeptide.

In accordance with still another embodiment of the present inventionthere are provided processes of administering compounds which bind toand inhibit activation of the receptor polypeptides of the presentinvention for prevention and/or treatment of conditions resulting fromexpression of the amine receptor of the present invention.

In accordance with yet another aspect of the present invention, thereare provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to the polynucleotidesequences of the present invention.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases related tomutations in the nucleic acid sequences encoding such polypeptides andfor detecting an altered level of the soluble form of the receptorpolypeptides.

In accordance with yet a further aspect of the present invention, thereare provided processes for utilizing such receptor polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 illustrates the cDNA sequence and corresponding deduced aminoacid sequence of the human amine receptor of the present invention. Thestandard one-letter abbreviations for amino acids are used. Sequencingwas performed using a 373 Automated DNA sequencer (Applied Biosystems,Inc.).

FIG. 2 is an illustration of an amino acid homology alignment betweenthe amine transporter or the present invention (top line) and murine β-1Adrenoreceptor (bottom line).

FIG. 3 is an illustration of an amino acid homology alignment betweenthe amine transporter or the present invention (top line) and humandopamine D2 receptor (bottom line).

The amine receptor of the present invention may be responsible forre-uptake of one or any of the amine neurotransmitters present inmammalian cells. Examples of such amine transporters include, but arenot limited to, dopamine, norepinephrine, epinephrine, serotonin andhistamine, and other amino acid transmitters, including GABA, glycineand glutamate.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 (SEQ IDNO:2) or for the mature polypeptide encoded by the cDNA of the clonedeposited as ATCC Deposit No. ______ on Jun. 1, 1995.

A polynucleotide encoding a polypeptide of the present invention may befound in human monocytes. The polynucleotide of this invention wasdiscovered in a human genomic library. It is structurally related to theG protein-coupled receptor family. It contains an open reading frameencoding a protein of 337 amino acid residues. The protein exhibits thehighest degree of homology to a murine β-1 Adrenoreceptor with 32.099%identity and 55.864% similarity over a 330 amino acid stretch. Theprotein also exhibits homology to a human dopamine D₂ receptor with 32%identity and 58.333% similarity over a 312 amino acid stretch.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID NO:1) or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of FIG. 1 (SEQ IDNO:1) or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG. 1(SEQ ID NO:2) or for the mature polypeptide encoded by the depositedcDNA may include: only the coding sequence for the mature polypeptide;the coding sequence for the mature polypeptide and additional codingsequence; the coding sequence for the mature polypeptide (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5′ and/or 3′ of the coding sequence for the maturepolypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or thepolypeptide encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the depositedclone. As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The polynucleotides may also encode for a soluble form of the aminereceptor polypeptide which is the extracellular portion of thepolypeptide which has been cleaved from the TM and intracellular domainof the full-length polypeptide of the present invention.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s), i.e. function as a soluble amine receptor byretaining the ability to bind the ligands for the receptor even thoughthe polypeptide does not function as a membrane bound amine receptor,for example, by eliciting a second messenger response.

Alternatively, the polynucleotides may have at least 20 bases,preferably 30 bases and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which have anidentity thereto, as hereinabove described, and which may or may notretain activity. Such polynucleotides may be employed as probes for thepolynucleotide of SEQ ID NO: 1, or for variants thereof, for example,for recovery of the polynucleotide or as a diagnostic probe or as a PCRprimer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to a human amine receptorpolypeptide which has the deduced amino acid sequence of FIG. 1 (SEQ IDNo. 2) or which has the amino acid sequence encoded by the depositedcDNA, as well as fragments, analogs and derivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIG. 1 (SEQ ID No. 2) or that encoded by the depositedcDNA, means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide, i.e. functions as an aminereceptor, or retains the ability to bind the ligand for the receptoreven though the polypeptide does not function as a G-protein coupledreceptor, for example, a soluble form of the receptor.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNo. 2) or that encoded by the deposited cDNA may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol) or (iv) one in which the additional amino acids arefused to the mature polypeptide which are employed for purification ofthe mature polypeptide or (v) one in which a fragment of the polypeptideis soluble, i.e. not membrane bound, yet still binds ligands to themembrane bound receptor. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and to portions of such polypeptide with such portion of the polypeptidegenerally contains at least 30 amino acids and more preferably at least50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis, therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region “leader and trailer” as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf 9; animal cells such as CHO, HEK, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, PSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAB-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences. optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEMI (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HEK, HeLa and BHKcell lines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The human amine receptor polypeptide can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

Fragments of the full length human amine transporter gene may be used asa hybridization probe for a cDNA library to isolate the full length geneand to isolate other genes which have a high sequence similarity to thegene or similar biological activity. Probes of this type are at least 20bases, preferably at least 30 bases and most preferably at least 50bases or more. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete human amine transporter gene includingregulatory and promotor regions, exons, and introns. As an example of ascreen comprises isolating the coding region of the human aminetransporter gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

This invention provides a method for determining amine neurotransmitterswhich are transported by the human amine receptor of the presentinvention. An example of an assay which will identify theseneurotransmitters comprises infecting mammalian cells with recombinantvaccinia virus strain VTF-7 encoding a T7 RNA polymerase and followingsuch infection with liposome-mediated transfection with the aminereceptor gene of the present invention through the use of a vector, forexample, pBSSKII(−). Controlled transfections are also done withequivalent amounts of vector alone. Assays are performed eight hoursfollowing transfection in modified Krebs-Ringer-HEPES buffer. Cells arethen incubated with [³H] neurotransmitter (for example, GABA, dopamine,serotonin, etc.). Uptake is stopped by placing the cells on ice. Cellsare solubilized in one percent SDS, and the amount of radioactivityaccumulated is determined by liquid scintillation counting. Asignificant amount of uptake determines that the particularneurotransmitter is taken up by the human amine receptor of the presentinvention by determining background using control transfections withpBSSKII for each assay and subtracting the values obtained from thesignals determined for the specific amine neurotransmitters.

This invention also provides a method of detecting expression of theamine receptor of the present invention on the surface of a cell bydetecting the presence of mRNA coding for the amine receptor. Thismethod comprises obtaining total mRNA from the cell using methodswell-known in the art and contacting the mRNA so obtained with a nucleicacid probe of at least 10 nucleotides and which is capable ofspecifically hybridizing with a sequence included within the sequence ofa nucleic acid molecule encoding a human amine receptor of the presentinvention under hybridizing conditions, detecting the presence of mRNAhybridized to the probe, and thereby detecting the expression of theamine receptor by the cell. Hybridization of probes to target nucleicacid molecules such as mRNA molecules employs techniques well known inthe art. However, in one embodiment of this invention, nucleic acids areextracted by precipitation from lysed cells and the mRNA is isolatedfrom the extract using a column which binds the poly-A tails of the mRNAmolecules. The mRNA is then exposed to radioactively labelled probe on anitrocellulose membrane, and the probe hybridizes to and thereby labelscomplementary mRNA sequences. Binding may be detected by autoradiographyor scintillation counting. However, other methods for performing thesesteps are well known to those of skill in the art.

Alternatively, an antibody directed to the human amine receptor may beemployed under conditions permitting binding of the antibody to thetransporter, and detecting the presence of the receptor on the surfaceof the cell. Such a method may be employed for determining whether agiven cell is defective in expression of the amine receptor. Detectionmethods include fluorescent markers bound to the antibodies.

The invention also provides a method for determining whether a compoundnot known to be capable of specifically binding to a human aminereceptor can specifically bind to the human amine receptor, whichcomprises contacting a mammalian cell comprising a plasmid adapted forexpression in a mammalian cell which plasmid further comprises a DNAwhich expresses the amine receptor on the cell surface with the compoundunder conditions permitting binding of ligands known to bind to theamine receptor, detecting the presence of any compound bound to theamine receptor, the presence of bound compound indicating that thecompound is capable of specifically binding to the human amine receptor.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease.

The amine receptor of the present invention may be employed in a processfor screening for compounds which activate (agonists) or inhibitactivation (antagonists) of the receptor polypeptide of the presentinvention In general, such screening procedures involve providingappropriate cells which express the receptor polypeptide of the presentinvention on the surface thereof. Such cells include cells from mammals,yeast, drosophila or E. Coli. In particular, a polynucleotide encodingthe receptor of the present invention is employed to transfect cells tothereby express the amine receptor. The expressed receptor is thencontacted with a test compound to observe binding, stimulation orinhibition of a functional response.

One such screening procedure involves the use of melanophores which aretransfected to express the amine receptor of the present invention. Sucha screening technique is described in PCT WO 92/01810 published Feb. 6,1992.

Thus, for example, such assay may be employed for screening for acompound which inhibits activation of the receptor polypeptide of thepresent invention by contacting the melanophore cells which encode thereceptor with both the receptor ligand and a compound to be screened.Inhibition of the signal generated by the ligand indicates that acompound is a potential antagonist for the receptor, i.e., inhibitsactivation of the receptor.

The screen may be employed for determining a compound which activatesthe receptor by contacting such cells with compounds to be screened anddetermining whether such compound generates a signal, i.e., activatesthe receptor.

Other screening techniques include the use of cells which express theamine receptor (for example, transfected CHO cells) in a system whichmeasures extracellular pH changes caused by receptor activation, forexample, as described in Science, volume 246, pages 181-296 (October1989). For example, compounds may be contacted with a cell whichexpresses the receptor polypeptide of the present invention and a secondmessenger response, e.g. signal transduction or pH changes, may bemeasured to determine whether the potential compound activates orinhibits the receptor.

Another such screening technique involves introducing RNA encoding theamine receptor into xenopus oocytes to transiently express the receptor.The receptor oocytes may then be contacted with the receptor ligand anda compound to be screened, followed by detection of inhibition oractivation of a calcium signal in the case of screening for compoundswhich are thought to inhibit activation of the receptor.

Another screening technique involves expressing the amine receptor inwhich the receptor is linked to a phospholipase C or D. Asrepresentative examples of such cells, there may be mentionedendothelial cells, smooth muscle cells, embryonic kidney cells, etc. Thescreening may be accomplished as hereinabove described by detectingactivation of the receptor or inhibition of activation of the receptorfrom the phospholipase second signal.

Another method involves screening for compounds which inhibit activationof the receptor polypeptide of the present invention antagonists bydetermining inhibition of binding of labeled ligand to cells which havethe receptor on the surface thereof. Such a method involves transfectinga eukaryotic cell with DNA encoding the amine receptor such that thecell expresses the receptor on its surface and contacting the cell witha compound in the presence of a labeled form of a known ligand. Theligand can be labeled, e.g., by radioactivity. The amount of labeledligand bound to the receptors is measured, e.g., by measuringradioactivity of the receptors. If the compound binds to the receptor asdetermined by a reduction of labeled ligand which binds to thereceptors, the binding of labeled ligand to the receptor is inhibited.

Amine receptors are ubiquitous in the mammalian host and are responsiblefor many biological functions, including many pathologies. Accordingly,it is desirous to find compounds and drugs which stimulate the aminereceptor on the one hand and which can inhibit the function of a aminereceptor on the other hand.

Examples of compounds which bind to and inhibit the amine receptor ofthe present invention includes antibodies, or in some cases anoligopeptides, which bind to the amine receptor but do not elicit asecond messenger response such that the activity of the amine receptoris prevented. Antibodies include anti-idiotypic antibodies whichrecognize unique determinants generally associated with theantigen-binding site of an antibody.

Another example includes proteins which are closely related to theligand of the amine receptors, i.e. a fragment of the ligand, which haslost biological function and when binding to the amine receptor, elicitsno response.

An antisense construct prepared through the use of antisense technology,may be used to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes for the mature polypeptides ofthe present invention, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073. (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of the amine receptor. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of mRNA moleculesinto amine receptor (antisense—Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). The oligonucleotides described abovecan also be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of the amine receptor.

A small molecule which binds to the amine receptor, making itinaccessible to ligands such that normal biological activity isprevented, for example small peptides or peptide-like molecules, mayalso be used to inhibit activation of the receptor polypeptide of thepresent invention.

A soluble form of the amine receptor, e.g. a fragment of the receptor,may be used to inhibit activation of the receptor by binding to theligand to the receptor polypeptide of the present invention andpreventing the ligand from interacting with membrane bound aminereceptors.

This invention additionally provides a method of treating an abnormalcondition related to expression of the amine receptor of the presentinvention which comprises administering to a subject an inhibitorycompound as hereinabove described along with a pharmaceuticallyacceptable carrier in an amount effective to block bind to a human aminereceptor can specifically bind to the human amine receptor, whichcomprises contacting a mammalian cell comprising a plasmid adapted forexpression in a mammalian cell which plasmid further comprises a DNAwhich expresses the amine receptor on the cell surface with the compoundunder conditions permitting binding of ligands known to bind to theamine receptor, detecting the presence of any compound bound to theamine receptor, the presence of bound compound indicating that thecompound is capable of specifically binding to the human amine receptor.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease.

The amine receptor of the present invention may be employed in a processfor screening for compounds which activate (agonists) or inhibitactivation (antagonists) of the receptor polypeptide of the presentinvention In general, such screening procedures involve providingappropriate cells which express the receptor polypeptide of the presentinvention on the surface thereof. Such cells include cells from mammals,yeast, drosophila or E. Coli. In particular, a polynucleotide encodingthe receptor of the present invention is employed to transfect cells tothereby express the amine receptor. The invention also provides apharmaceutical pack or kit comprising one or more containers filled withone or more of the ingredients of the pharmaceutical compositions of theinvention. Associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In addition, the pharmaceutical compositions may beemployed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10 μg/kgbody weight and in most cases they will be administered in an amount notin excess of about 8 mg/Kg body weight per day. In most cases, thedosage is from about 10 μg/kg to about 1 mg/kg body weight daily, takinginto account the routes of administration, symptoms, etc.

The human amine receptor and agonist and antagonist compounds which arepolypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as—the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe genes encoding the polypeptides.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PES01, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPo₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the human amine receptorgene as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutations in thehuman amine receptor genes. Such diseases are related tounder-expression of the human amine receptor.

Individuals carrying mutations in the human amine receptor gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding the human aminereceptor protein can be used to identify and analyze human aminereceptor mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled human amine receptor RNA or alternatively,radiolabeled human amine receptor antisense DNA sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNaseA digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of soluble forms of the amine receptor polypeptides ofthe present invention in various tissues which may be employed todiagnose diseases related to under-expression of the amine receptor.Assays used to detect levels of the soluble receptor polypeptides in asample derived from a host are well known to those of skill in the artand include radioimmunoassays, competitive-binding assays, Western blotanalysis and preferably as ELISA assay.

An ELISA assay initially comprises preparing an antibody specific toantigens of the amine receptor polypeptides, preferably a monoclonalantibody. In addition a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as radioactivity, fluorescence or in this example ahorseradish peroxidase enzyme. A sample is now removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein such as bovineserum albumin. Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any amine receptorproteins attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is now placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to amine receptorproteins. Unattached reporter antibody is then washed out. Peroxidasesubstrates are then added to the dish and the amount of color developedin a given time period is a measurement of the amount of amine receptorproteins present in a given volume of patient sample when comparedagainst a standard curve.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 50 or 60bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide orpreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available-plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Purification of Human Amine Receptor

The DNA sequence encoding human amine receptor, ATCC # ______, isinitially amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ end sequences of the processed amine receptor nucleic acidsequence (minus the signal peptide sequence). Additional nucleotidescorresponding to amine receptor gene are added to the 5′ and 3′sequences respectively. The 5′ oligonucleotide primer has the sequence5′ CGGAATTCCTUATGAGAGCTGTCTTCATC 3′ (SEQ ID No. 3) contains an EcoRIrestriction enzyme site followed by 18 nucleotides of human aminereceptor coding sequence starting from the presumed terminal amino acidof the processed protein. The 3′ sequence 5′CGGAAGCTTCGTCATTCTTGGTACAAATCAAC 3′ (SEQ ID No. 4) containscomplementary sequences to an HindIII site and is followed by 18nucleotides of the human amine receptor gene. The restriction enzymesites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/o), a ribosome bindingsite (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is thendigested with HindIII and EcoRI. The amplified sequences are ligatedinto pQE-9 and are inserted in frame with the sequence encoding for thehistidine tag and the RBS. The ligation mixture is then used totransform E. coli strain M15/rep 4 (Qiagen, Inc.) by the proceduredescribed in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiplecopies of the plasmid pREP4, which expresses the laci repressor and alsoconfers kanamycin resistance (Kan^(r)). Transformants are identified bytheir ability to grow on LB plates and ampicillin/kanamycin resistantcolonies are selected. Plasmid DNA is isolated and confirmed byrestriction analysis. Clones containing the desired constructs are grownovernight (O/N) in liquid culture in LB media supplemented with both Amp(100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate alarge culture at a ratio of 1:100 to 1:250. The cells are grown to anoptical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells are grownan extra 3 to 4 hours. Cells are then harvested by centrifugation.. Thecell pellet is solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized human amine receptor is purifiedfrom this solution by chromatography on a Nickel-Chelate column underconditions that allow for tight binding by proteins containing the 6-Histag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). Humanamine receptor protein is eluted from the column in 6 molar guanidineHCl pH 5.0 and for the purpose of renaturation adjusted to 3 molarguanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione (reduced)and 2 mmolar glutathione (oxidized). After incubation in this solutionfor 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2 Cloning and Expression of Human Amine Receptor Using theBaculovirus Expression System

The DNA sequence encoding the full length human amine receptor protein,ATCC # ______, is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

The 5′ primer has the sequence 5′5′ CGGGATCCCTCCATGAGA GCTGTCTTCATC 3′(SEQ ID No. 5) and contains a BamHI restriction enzyme site followed by4 nucleotides resembling an efficient signal for the initiation oftranslation in eukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950(1987) which is just behind the first 18 nucleotides of the human aminereceptor gene.

The 3′ primer has the sequence 5′ CGGGATCCCGCTCATTCTTGG TACAAATC 3′ (SEQID No. 6) and contains the cleavage site for the restrictionendonuclease BamHI and 18 nucleotides complementary to the 3′non-translated sequence of the human amine receptor gene. The amplifiedsequences are isolated from a 1% agarose gel using a commerciallyavailable kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). Thefragment is then digested with the endonucleases BamHI and then purifiedagain on a 1% agarose gel. This fragment is designated F2.

The vector PRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the human amine receptor protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin,No. 1555). This expression vector contains the strong polyhedrinpromoter of the Autographa californica nuclear polyhedrosis virus(AcMNPV) followed by the recognition sites for the restrictionendonucleases BamHI. The polyadenylation site of the simian virus (SV)40is used for efficient polyadenylation. For an easy selection ofrecombinant viruses the beta-galactosidase gene from E. coli is insertedin the same orientation as the polyhedrin promoter followed by thepolyadenylation signal of the polyhedrin gene. The polyhedrin sequencesare flanked at both sides by viral sequences for the cell-mediatedhomologous recombination of co-transfected wild-type viral DNA. Manyother baculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIMI (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes BamHI and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA is then isolated from a 1% agarose gel using thecommercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E. coli HB101 cells are then transformed and bacteria identifiedthat contained the plasmid (pBac-Human amine receptor) with the humanamine receptor gene using the enzyme BamHI. The sequence of the clonedfragment is confirmed by DNA sequencing.

5 μg of the plasmid pBac-Human amine receptor is co-transfected with 1.0μg of a commercially available linearized baculovirus (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofectionmethod (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac-Human aminereceptor are mixed in a sterile well of a microtiter plate containing 50μl of serum free Grace's medium (Life Technologies Inc., Gaithersburg,Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added,mixed and incubated for 15 minutes at room temperature. Then thetransfection mixture is added drop-wise to the Sf9 insect cells (ATCCCRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace'smedium without serum. The plate is rocked back and forth to mix thenewly added solution. The plate is then incubated for 5 hours at 27° C.After 5 hours the transfection solution is removed from the plate and 1ml of Grace's insect medium supplemented with 10% fetal calf serum isadded. The plate is put back into an incubator and cultivation continuedat 27° C. for four days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue-Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses are added to the cellsand blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-Human amine receptor at a multiplicity of infection (MOI)of 2. Six hours later the medium is removed and replaced with SF900 IImedium minus methionine and cysteine (Life Technologies Inc.,Gaithersburg). 42 hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵Scysteine (Amersham) are added. The cells are further incubated for 16hours before they are harvested by centrifugation and the labelledproteins visualized by SDS-PAGE and autoradiography.

EXAMPLE 3 Expression of Recombinant Human Amine Receptor in COS Cells

The expression of plasmid, Human amine receptor HA is derived from avector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin ofreplication, 2) ampicillin resistance gene, 3) E. coli replicationorigin, 4) CMV promoter followed by a polylinker region, a SV40 intronand polyadenylation site. A DNA fragment encoding the entire Human aminereceptor precursor and a HA tag fused in frame to its 3′ end is clonedinto the polylinker region of the vector, therefore, the recombinantprotein expression is directed under the CMV promoter. The HA tagcorrespond to an epitope derived from the influenza hemagglutininprotein as previously described (I. Wilson, et al., Cell, 37:767,(1984)). The infusion of HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding Human amine receptor, ATCC # ______, isconstructed by PCR using two primers: the 5′ primer 5′GTCCAAGCTTGCCACCATGAGAGCTGTCTTCATC.3′ (SEQ ID No. 7) contains a HindIIIsite followed by 18 nucleotides of Human amine receptor coding sequencestarting from the initiation codon; the 3′ sequence 5′CTAGCTCGAGTCAAGCGTA GTCTGGGACGTCGTATGGGTAGCATTCTTGGTACAAATCAAC 3′ (SEQID No. 8) contains complementary sequences to an XhoI site, translationstop codon, HA tag and the last 18 nucleotides of the Human aminereceptor coding sequence (not including the stop codon). Therefore, thePCR product contains a HindIII site, human amine receptor codingsequence followed by HA tag fused in frame, a translation terminationstop codon next to the HA tag, and an HindIII site. The PCR amplifiedDNA fragment and the vector, pcDNAI/Amp, are digested with HindIII andXhoI restriction enzymes and ligated. The ligation mixture istransformed into E. coli strain SURE (Stratagene Cloning Systems, LaJolla, Calif.) the transformed culture is plated on ampicillin mediaplates and resistant colonies are selected. Plasmid DNA is isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant amine receptor,COS cells are transfected with the expression vector by DEAE-DEXTRANmethod (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989)). The expressionof the Human amine receptor HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with ³⁵S-cysteine two days post transfection. Culture mediais then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5)(Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culturemedia are precipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 4 Expression Pattern of Human Amine Receptor in Human Tissue

Northern blot analysis is carried out to examine the levels ofexpression of Human amine receptor in human tissues. Total cellular RNAsamples are isolated with RNAzol™ B system (Biotecx Laboratories, Inc.Houston, Tex.) About 10 μg of total RNA isolated from each human tissuespecified is separated on 1% agarose gel and blotted onto a nylon filter(Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring HarborPress, (1989)). The labeling reaction is done according to theStratagene Prime-It kit with 50 ng DNA fragment. The labeled DNA ispurified with a Select-G-50 column (5 Prime-3 Prime, Inc. Boulder,Colo.). The filter is then hybridized with radioactive labeled fulllength Human amine receptor gene at 1,000,000 cpm/ml in 0.5 M NaPO₄, pH7.4 and 7% SDS overnight at 65° C. After wash twice at room temperatureand twice at 60° C. with 0.5×SSC, 0.1% SDS, the filter is then exposedat −70° C. overnight with an intensifying screen.

EXAMPLE 5 Expression Via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer contains an EcoRI site and the 3′ primercontains a HindIII site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the EcoRI and HindIII fragment are addedtogether, in the presence of T4 DNA ligase. The resulting mixture ismaintained under conditions appropriate for ligation of the twofragments. The ligation mixture is used to transform bacteria HB101,which are then plated onto agar-containing kanamycin for the purpose ofconfirming that the vector had the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1. An isolated polynucleotide comprising a member selected from thegroup consisting of: (a) a polynucleotide encoding the polypeptide asset forth in FIG.
 1. (b) a polynucleotide encoding the polypeptideexpressed by the DNA contained in ATCC Deposit No. ______; (c) apolynucleotide capable of hybridizing to and which is at least 70%identical to the polynucleotide of (a) or (b); and (d) a polynucleotidefragment of the polynucleotide of (a), (b) or (c).
 2. The polynucleotideof claim 1 encoding the polypeptide of FIG.
 1. 3. The polynucleotide ofclaim 1 wherein said polynucleotide encodes a mature polypeptide encodedby the DNA contained in ATCC Deposit No. ______.
 4. A vector containingthe polynucleotide of claim
 1. 5. A host cell genetically engineeredwith the vector of claim
 4. 6. A process for producing a polypeptidecomprising: expressing from the host cell of claim 5 the polypeptideencoded by said polynucleotide.
 7. A process for producing cells capableof expressing a polypeptide comprising genetically engineering cellswith the vector of claim
 4. 8. A polypeptide selected from the groupconsisting of (i) a polypeptide having the deduced amino acid sequenceof FIG. 1 and fragments, analogs and derivatives thereof; and (ii) apolypeptide encoded by the cDNA of ATCC Deposit No. ______ andfragments, analogs and derivatives of said polypeptide.
 9. Thepolypeptide of claim 8 wherein the polypeptide has the deduced aminoacid sequence of FIG.
 1. 10. An antibody against the polypeptide ofclaim
 8. 11. A compound which activates the polypeptide of claim
 8. 12.A compound which inhibits activation of the polypeptide of claim
 8. 13.A method for the treatment of a patient having need to activate areceptor comprising: administering to the patient a therapeuticallyeffective amount of the compound of claim
 11. 14. A method for thetreatment of a patient having need to inhibit a receptor comprising:administering to the patient a therapeutically effective amount of thecompound of claim
 12. 15. The method of claim 13 wherein said compoundis a polypeptide and a therapeutically effective amount of the compoundis administered by providing to the patient DNA encoding said agonistand expressing said agonist in vivo.
 16. The method of claim 14 whereinsaid compound is a polypeptide and a therapeutically effective amount ofthe compound is administered by providing to the patient DNA encodingsaid antagonist and expressing said antagonist in vivo.
 17. A method foridentifying a compound which bind to and activate the polypeptide ofclaim 8 comprising: contacting a compound with cells expressing on thesurface thereof the polypeptide of claim 8, said polypeptide beingassociated with a second component capable of providing a detectablesignal in response to the binding of a compound to said polypeptide saidcontacting being under conditions sufficient to permit binding ofcompounds to the polypeptide; and identifying a compound capable ofpolypeptide binding by detecting the signal produced by said secondcomponent.
 18. A method for identifying compounds which bind to andinhibit activation of the polypeptide of claim 8 comprising: contactingan analytically detectable ligand known to bind to the receptorpolypeptide and a compound with host cells expressing on the surfacethereof the polypeptide of claim 8, said polypeptide being associatedwith a second component capable of providing a detectable signal inresponse to the binding of a compound to said polypeptide underconditions to permit binding to the polypeptide; and determining whetherthe ligand binds to the polypeptide by detecting the absence of a signalgenerated from the interaction of the ligand with the polypeptide.
 19. Aprocess for diagnosing in a patient a disease or a susceptibility to adisease related to an under-expression of the polypeptide of claim 8comprising: determining a mutation in the nucleic acid sequence encodingsaid polypeptide, or the amount of the polypeptide in a sample derivedfrom a patient.
 20. A diagnostic process comprising: analyzing for thepresence of a soluble form of the polypeptide of claim 8 in a samplederived from a host.